During gestation, transport by placental trophoblasts is solely responsible for nutrient supply to the developing fetus. The Ca transport machinery of the placenta thus represents the primary tissue site for regulating fetal Ca homeostasis. The vital role of Ca in a multitude of biological functions, including skeletal formation, neuromuscular and cardiovascular activities, etc., as well as cellular signal transduction, underscores the importance of understanding the cellular and molecular mechanisms of placental Ca transport. Previous and current studies by this investigator have focused on elucidating the functional components of the placental Ca transport machinery. Specifically, a 1,25(OH)2 vitamin D3-dependent, high-M, Ca-binding protein (CaBP) has been identified and cloned, and localized to fetal placental trophoblasts. More recently, we have constructed an in vitro cellular model system, using the human choriocarcinoma cell line, JEG-3, to study trophoblast transcellular Ca transport; our findings showed that this system responds to 1,25(OH)2 vitamin D3 with enhancement of both CaBP expression and Ca uptake. These findings serve as the basis of the experimental plan proposed here. Specific Aim 1. Cellular mechanism of placental Ca Transport. This will involve validating and exploiting the in vitro Ca transport system, a trophoblastic epithelium formed with JEG- 3 cultured on a permeable membrane, to analyze transcellular Ca transport with respect to two possible pathways (intracellular versus transcytotic) and the functional role of the CaBP. Specific Aim 2. Regulation of CaBP expression. The spatiotemporal profiles of CaBP expression in the placenta during development will be characterized, and the molecular steps resulting in vitamin D-mediated up-regulation of CaBP expression will be examined. Specific Aim 3. Molecular Mechanism of Cd toxicity on placental Ca transport. The role of metallothionein will be analyzed in order to localize the "site of action" of Cd-mediated poisoning. Our overall approach to studying placental Ca transport is thus an integrated one, such that the transport function is experimentally reconstituted, dissected and modified to probe its cellular, biochemical, and molecular regulatory aspects, including the mechanistic basis of heavy metal poisoning. Results from these studies should enhance our understanding of an important nutritional and physiological function during fetal development, and may also provide insights into epithelial transport processes in general. Finally, we hope that, in the long term, our studies can contribute to understanding how the inappropriate regulation and/or perturbation of placental Ca transport may give rise to various maternal and fetal dysfunctions, including intrauterine growth retardation.